HPLC Troubleshooting Guide

Troubleshooting in
HPLC analysis
Spinco Biotech Pvt. Ltd.

General Troubleshooting
Troubleshooting
 Mobile phase
 Sample
 Injection
 Column
 Detection
Mobile phase Solvent
Grade and Treatment
Absorptio
deionized
Water grade water
Pure water : Organic and Ion pure
n
Distilled water : Organic water
Deionized water : Ion Wavelength (nm)
Millipore or Barnstead system is ideal as they remove inorganic and

organic contaminants.
Modern pure water systems give TOC values as indication of
total organic content in water.
Solvent Grade and Treatment
Difference of Analytical and HPLC Grade
Methanol Acetonitrile Hexane

Ghost peak will be appeared in gradient elution if water is

of bad quality!
H2O / MeOH r ve
Cu
gradient nt
ie
r ad
G
ODS Column
1 ml/min, 0-100% MeOH

over 10 mins and hold for
15 mins. Ghost Peaks
HPLC Grade
HPLC Grade solvents only guarantee low UV

absorbing impurities.
It does not mean it is free of all impurities.
-- All solvents to be used should be filtered through at least

0.45um membrane filters before use.
If the solvent has background absorption,
-- linear range will be smaller,

-- noise will be bigger and
-- baseline stability especially during gradient
runs will be poor.
Some precautions in handling HPLC Grade organic solvents :
1. THF
Analytical grade THF,

contains butylated hydroxytoluene(BHT) as an oxidation
inhibitor. BHT has strong UV absorption.
HPLC grade THF,

It does not contain BHT.
HPLC grade THF must be stored in cool and dark
places to reduce oxidation to form peroxides.
Accumulation of large amount of organic peroxides may

lead to explosions.
2. Chloroform
Analytical grade chloroform contains 3% ethanol to

prevent generation of phosgene, a highly toxic gas.
However, ethanol, like water, may deactivate silica gel used
in normal phase separation and thus lead to unstable
retention times.
HPLC Grade chloroform therefore does not contain ethanol

as a stabiliser. Once seal is opened, it should be stored in
dark and cool places to reduce the possibility of generation
of phosgene.
Use of Buffer Solutions
Solutions must be filtered before use.

Must not be left in system to avoid crystallization :
 effect on pump -- damage plunger and seal
 effect on column -- creation of column voids
 effect on flow-line -- corrosion of stainless steel lines
Possible bacterial growth
especially phosphate buffers -- good medium
for bacterial and fungus growth.
Ideally, solutions should be prepared fresh everyday.

Maintenance and
Troubleshooting Solvent Grade and Treatment
Solvent Filtration Kit

Changing Over of Mobile Phase
Buffer solution
Water
Aqueous Organic Solvent

( methanol, acetonitrile etc. )
2-propanol
Non-aqueous Organic Solvent

( hexane, chloroform )
Pipe
Do not
20 cm
touch
directly
Suction Filter
rinse suction filter
Maintenance and
Troubleshooting Solvent Grade and Treatment
Premixing of Solvents
For isocratic systems, we often premixed the solvent mixtures
used for analysis.
E.g. To prepare 100ml of 50:50 mixture of water/methanol,
we may :
1. Measure out 50ml of water and 50ml of methanol separately
then mix them together.
2. Measure 50 ml of water and top up to 100ml mark of
measuring cylinder/volumetric flask with methanol.
3. Measure 50ml of methanol and top up to 100ml with water.
All three methods will give different compositions. Whichever

method selected must be used consistently for repeatable results.
Solvent Reservoir Problems
1. Containers should generally be glass as plastics will

leach plasticers leading to unstable baseline and/or
contamination of columns.
Glass should be avoided when carrying out high sensitivity
analysis if inorganic ions as these could be leached from
glass.
2. Containers should have small covered openings. This is to

prevent rapid evaporation of solvents leading to unstable
compositions. It also serves to reduce the vapours in the
room. Do not make containers airtight as this may lead
to creation of partial vacuum and pump starvation.
3. When using stabiliser free solvents, either change fresh

solvents daily or provide inert atmosphere for solvent
reservoir such as use of helium degasser.
4. Do not position solvents directly in line of drafts from

ventilation ducts, windows, doors or hoods; do not position
where it may be exposed to direct sunlight. This may lead
to temperature gradients and/or increased evaporation.
Effect will be greatest for detectors like RI detectors but
sometimes may also affect UV detectors.
5. Always keep reservoir above solvent delivery system as it

ensures good siphon feed to pump and prevents starving
pumping system.
6. Always use a suction filter. This is course filter for solvent

and helps to prevent particulates from reaching pumps.
Keep these filters clean and free from fungus. If dirty,
they may be cleaned by sonicating in beaker of isopropyl
alcohol or 1N nitric acid.
7. Good practice in the lab to label all bottles with composition

inside as well as date of preparation.
Sample Treatment Problems
1. It is always best to dissolve sample in mobile phase.

-- reduces solvent peak and is important in cases
where peak of interest elute close to solvent peak.
-- ensures solubility of sample in mobile phase so it
does not precipitate in the system, especially in column.
-- ensures miscibility of sample solvent with mobile
phase.
2. Always filter sample with 0.45um filters before injection.

If sample is known to be very dirty, filter through 0.2um
filters before injection.
The cause of quantitative error for

sample pretreatment
 Standard solution
 Measure weight
 Accuracy of balance
 Operation
 Impurity
 Uncertain hydrate number
 Hygroscopic

sample pretreatment
 Measure up by solvent
 Accuracy of apparatuses
 Solubility(temp.)
 Inhomogeneous
 Adsorption( to apparatus)
 E.t.c.

sample pretreatment
 Storage
 Evaporation
 Oxidation
 decomposition
Adsorption of target compounds

on the apparatus
Material Compounds
Glass Cation[Quarternary Decrease pH
ammonium, Metal ions] Add ClO4 ion
Amine
Metal Anion Decrease pH

Chelate compounds
Polymer Hydrophobic
compounds
Injector
Loop type Injector
Two injection methods are possible :
1. Partial Loop Injection Method

-- Inject less than half volume of sample loop.
2. Full Loop Injection Method

-- Inject more than 3 times volume of sample loop.
Injector
Loop type Injector

Relationship between injection volume and detector's response
Response of Detector
Loop injection
Partial
injection
3 times volume
Half volume
of sample loop
of sample loop
Volume of Injection
Injector
Manual Injector -- Rheodyne

Results can be explained in terms of Dispersion and
Dilution effects.
Less than half volume More than half volume
dr dr
a in p a in p
m m
pu p u
co co
lu lu
m m
in n n
r a in
d a
dr
loop loop
Sample Zone
Dilution Zone
Dispersion Zone
Injector
Manual Injector -- Rheodyne
Less than 3 times More than 3 times

dr p dr
a in m a p
pu in m
pu
co co
lu lu
m m
n n
ain a in
dr dr
loop loop
Sample Zone
Dilution Zone
Injector
Auto-Injectors -- General Maintenance
1. Purge flow line at least once a day.
2. Change purge liquid at least once a week.
3. Never use salt solutions as purge liquid.
4. Use only septums recommended by manufacturer.
5. Septums should not be reused.
6. Do not fill vials to the brim.

Auto-injector SIL-10ADVP
27
SIL-10ADvp/LC-2010
Sample Loop Sample Loop

Rinse Rinse
Port Port
Pump Pump
INJ Port INJ Port
Column Column
READY Mode Auto Drain Mode

Column
Initial checks on new columns.
1. Column performance check
Column performance check may be carried out before
column is first used using the test mixture and condition
as stipulated in the column performance sheet.
2. Pressure check
Initial operating pressure of the column under normal
analysis conditions should be noted for reference.
3. Guard Columns
Should be used whenever possible but should not add much
pressure to system.
Column
Precautions During Operation.

1. For new columns, it is best to condition under low
flow rate (0.2-0.3 ml/min) for about 30 mins before operating
under analytical flow rate. Same applies for columns that
have not been used for long time.
2. During shutdown, it is best to gradually reduce flow 　 rate so as

not to subject column to sudden pressure drop.
3. When using buffer solutions, remember to flush with water

before changing to organic solvents to prevent crystallization.
Column
4. It is best to filter mobile phase and sample before use to

reduce clogging by particulates in frit.
5. Sample is best dissolved in mobile phase or if not possible

then as close to mobile phase composition as possible.
This reduce chances that sample may precipitate in column
bed on mixing with mobile phase.
6. Flush with stronger solvent after daily use. For example,

ODS columns may be flushed with acetonitrile after use
daily to remove strongly absorbed materials from column.
Column
Column Troubles and Solutions
High pressure in system

Poor peak shape (tailing, leading, splitting)
Changes in retention time.
Plugged Frit or Column Packing

Adsorbed Sample & Solvent Impurities
Voids Resulting from Shock or Crystallization
Chemical Attack of Packing Material
Drying up of Packing Material
Column
Column Problems and Solutions -- Plugged Frit or Column Packing
Prevention is by filtering both sample and mobile phase

through 0.45m membrane filter or 0.2m membrane
filter if sample is very dirty.
However, sometimes, precipitation may occur or some dirt

may get into system due to exposure of mobile phase or sample
and this will result in clogging.
Column
Column Problems and Solutions -- Plugged Frit or Column Packing
Normal flushing with string solvent may dissolve and remove

some of these particles but in cases where the particulates are
insoluble, flushing the column in reverse may help.
Note : Do not connect to detector in this case and do not use

this method for routine flushing as it may lead to loosening of
packing material.
Finally, replace filter frit if it cannot be washed. Be careful

not to disturb column bed or performance may drop and
in some cases, the stainless steel frits are fused to the columns
and these cannot be replaced.
Column
Column Problems and Solutions --
Adsorbed Sample & Solvent Impurities
Impurities will cause a tailing peak.

Wash a column using high solubility solvent.
Remove this parts and

repack the packing material.
only 1 cm will
column
be polluted
LC
Column
As HPLC columns are pressure-packed, subjecting them to

pressure shock such as dropping the column or opening of
drain valve during operation may result in formation of voids.
In the event of crystallization, even though the crystals may be

Re-dissolved and was away, the packing pushed aside by their
presence may not return to original place resulting in voids.
Do not open drain valve

LC-10AD :::::::
during operation
000 0 000 0
xxxx xxxx
7 8 9
xxxx xxxx 4 5 6
xxxx xxxx
1 2 3
xxxx xxxx
0 ¥ xxxx
Column

Voids will cause split peaks.
void
Am I seeing
Every peak will be split. double?
Hard to repair!!!
Column
Chemical Attack of Packing Material
Silica gel Polymer

pH range 2 - 7.5 widely use
Organic solvent all solvent limitation
Pressure less than 250 kgf/cm2 low pressure
Temperature better to set possible to set
at less than 60C at high temperature
Caution : Must check the value in terms of

every item of polymer based column
Column

Drying up of Packing Material
Frequently, especially for polymer based columns, swelling

characteristics (shape) of packing depends very much on
solvent. When column is dry, these may be deformed
permanently.
Especially for GPC type columns, the choice of solvent is

used is very specific. Not only must it be kept wet, it must
not be used with wrong type of solvent or whole characteristic
of polymer (shape and pore size) will be changed permanently.
Waste Bottle
Make sure that waste line is not immersed in liquid waste

in
the waste bottle as this may create back pressure on cell.
Detector Detector
Detector cell
Back pressure
0.3mmI.D. x 2mL.
Maintenance and
Troubleshooting
Other Trouble
Peak Shape Distortion -- A. Asymmetry (Leading / Tailing)
Case 1 : development of method

Possible causes : 1. Improper solvent selection (may also result in
peak splitting
Methanol as a sample solvent Ethanol as a sample solvent
20 L Caffeine 20 L Caffeine
Ethanol as a sample solvent Better inject small

amount of sample.
10 L Caffeine
Maintenance and
Troubleshooting
Other Trouble

SOLUBILITY EFFECT
Low soluble solvent High soluble solvent

Maintenance and
Troubleshooting
Other Trouble
2. Secondary retention effects : due to heavy metals in silica gel or
residual silanol groups in modified silica gel.
C18
OH Silanol group
C18
silica core
-
OC18 negative charge
C18
C18
C18
OH O-TMS
silica core C18 TMS treatment C18
OH silica core O-TMS
C18 TMS : trimethylsilyl group
C18
C18
C18 [End capping type]
[Non-End capping type]
Maintenance and
Troubleshooting
Other Trouble
C18
+ O-TMS High pure type of
silicaMcore C18 O silica gel are available.
O-TMS
M+ C18 O
C18
3. Overloading of sample
For every column, no. of binding sites is limited, if sample loading grossly
exceeds the columns capacity, leading or tailing may occur (most probably
tailing).
4. Adjustment of pH of mobile phase.

Maintenance and
Troubleshooting
Other Trouble
Case 2 : Sudden peak shape distortions in existing methods
1. Column (Guard/Main) failure. -- clean or replace column.

2. Column dissolution may occur for example if high pH (>7) is used with
silica based column.
3. Problems with secondary retention effects may occur if different make
of column is used.
4. In particular, broadening may be caused by dead volumes.
Good
Dead Volume

HPLC Troubleshooting Guide

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Troubleshooting in

Spinco Biotech Pvt. Ltd.

Millipore or Barnstead system is ideal as they remove inorganic and

Difference of Analytical and HPLC Grade

Methanol Acetonitrile Hexane

Ghost peak will be appeared in gradient elution if water is

1 ml/min, 0-100% MeOH

HPLC Grade solvents only guarantee low UV

-- All solvents to be used should be filtered through at least

-- linear range will be smaller,

Some precautions in handling HPLC Grade organic solvents :

Analytical grade THF,

HPLC grade THF,

Accumulation of large amount of organic peroxides may

Analytical grade chloroform contains 3% ethanol to

HPLC Grade chloroform therefore does not contain ethanol

Use of Buffer Solutions

Solutions must be filtered before use.

Ideally, solutions should be prepared fresh everyday.

Solvent Filtration Kit

Changing Over of Mobile Phase

Aqueous Organic Solvent

Non-aqueous Organic Solvent

All three methods will give different compositions. Whichever

1. Containers should generally be glass as plastics will

2. Containers should have small covered openings. This is to

3. When using stabiliser free solvents, either change fresh

4. Do not position solvents directly in line of drafts from

5. Always keep reservoir above solvent delivery system as it

6. Always use a suction filter. This is course filter for solvent

7. Good practice in the lab to label all bottles with composition

1. It is always best to dissolve sample in mobile phase.

2. Always filter sample with 0.45um filters before injection.

The cause of quantitative error for

 Uncertain hydrate number

The cause of quantitative error for

The cause of quantitative error for

Adsorption of target compounds

Metal Anion Decrease pH

Loop type Injector

Two injection methods are possible :

1. Partial Loop Injection Method

2. Full Loop Injection Method

Loop type Injector

Manual Injector -- Rheodyne

Manual Injector -- Rheodyne

Less than 3 times More than 3 times

Auto-Injectors -- General Maintenance

1. Purge flow line at least once a day.

2. Change purge liquid at least once a week.

3. Never use salt solutions as purge liquid.

4. Use only septums recommended by manufacturer.

5. Septums should not be reused.

6. Do not fill vials to the brim.

Sample Loop Sample Loop

INJ Port INJ Port

READY Mode Auto Drain Mode

Precautions During Operation.

2. During shutdown, it is best to gradually reduce flow rate so as

3. When using buffer solutions, remember to flush with water

4. It is best to filter mobile phase and sample before use to

5. Sample is best dissolved in mobile phase or if not possible

2. During shutdown, it is best to gradually reduce flow 　 rate so as